Seismic exploration at Fuji volcano with active sources : The outline of the experiment and the arrival time data

Fuji volcano (altitude 3,776m) is the largest basaltic stratovolcano in Japan. In late August and early September 2003, seismic exploration was conducted around Fuji volcano by the detonation of 500 kg charges of dynamite to investigate the seismic structure of that area. Seismographs with an eigenfrequency of 2 Hz were used for observation, positioned along a WSW-ENE line passing through the summit of the mountain. A total of 469 seismic stations were installed at intervals of 250-500 m. The data were stored in memory on-site using data loggers. The sampling interval was 4 ms. Charges were detonated at 5 points, one at each end of the observation line and 3 along its length. The first arrival times and the later-phase arrival times at each station for each detonation were recorded as data. P-wave velocities in the surface layer were estimated from the travel time curves near the explosion points, with results of 2.5 km/s obtained for the vicinity of Fuji volcano and 4.0 km5/s elsewhere

Non-specific protein modifications by a phytochemical induce heat shock response for self-defense.

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities

Proposed mechanisms underlying HSP70 induction by ZER.

<p>Xenobiotic phytochemicals like ZER are bound to numerous cellular proteins with less selectivities, which are recognized as proteostress by HSP90. Subsequently, HSF1 was dissociated from HSP90 and activated for HSPs induction.</p

ZER induced HSPs expressions and conferred thermoresistant phenotype <i>in vivo</i>.

<p>(A) Hepa1c1c7 cells were treated with ZER (0, 10, 25, 50 µM) for 24 hours or exposed to mild heat (43°C) for 1 hour (HT), followed by recovery at 37°C for 6 hours. Cells were then exposed to heat shock (45°C) for 1 hour. After another 12 hours of incubation, cell viability was determined using a WST-8 test. Non-treated cells were used as a negative control (NT). This experiment was performed in triplicates. (B) D3 nematode larvae were pretreated with ZER (100, 200, 400 µM) for 3 days or pre-exposed to mild heat (33°C) for 1 hour (HT), followed by recovery at 25°C for 24 hours. After exposure to heat shock (incubation in 37°C waterbath) for 1 hour, survival rate was determined. Non-treated nematodes (NT) were used as a negative control. This experiment was performed in quadruplicates. The various characteristics were significantly different, as shown by Tukey-Kramer test results (<i>P</i><0.05) (A, B). (C) D3 larvae nematodes were treated with ZER (Z; 100–400 µM) or the vehicle (D; 0.5% DMSO, <i>v/v</i>) for 62 hours, or exposed to mild heat (33°C) for 1 hour (H), followed by recovery at 25°C for 24 hours. HSPs expressions were semi-quantified by qRT-PCR. CDC42 expressions were also measured as internal standards. a, versus CTL; b, versus DMSO by Dunnett's test (<i>P</i><0.05). This experiment was performed in quintuplicate. (D) SD rats (8 weeks old) were orally administered ZER (50 mg/kg) or the vehicle (CTL, corn oil) twice a day for 1 week, then euthanized and the livers were collected. Those were lysed with PBS, then lysates were added to NacZER-BSA-coated ELISA places as a competitor with a thioether ZER Ab solution. The amount of ZER-thiol adducts in liver lysates was semi-quantified based on a calibration curve using NacZER. This experiment was performed in triplicates. (E) Livers lysates were also subjected to western blot analysis for HSP70 determination. This experiment was performed in sextuplicate.*<i>P</i><0.05 vs. CTL by Student's <i>t</i> test (D, E).</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Biotin derivative of ZER was bound to numerous proteins with less selectivities.

<p>(A) BrZER was chemically reacted with biotin to yield the biotin derivative of ZER (BioZER). (B) Hepa1c1c7 cells were pretreated with the vehicle, ZER, or BioZER (0.8–20 µM), then exposed to LPS (100 ng/mL) for 24 hours. NO generation was quantified by measuring the concentration of nitrite using Griess reagent. The various characteristics were significantly different, as shown by Tukey-Kramer test results (<i>P</i><0.05). This experiment was performed in triplicates. (C) Cells were treated with BioZER (10 µM) for 0 minutes to 6 hours, then washed with PBS and incubation in BioZER-free DMEM for another 1–42 hours. Cells were lysed for detection of modified proteins by western blot analysis with HRP-conjugated avidin. (D) Cells were co-treated with the vehicle or BioZER (1, 10 µM) with or without ZER (100 µM) for 30 minutes, then lysed for detection of modified proteins using western blot analysis with HRP-conjugated avidin.</p

ZER induced HSPs expressions through HSF1 activation.

<p>(A) Hepa1c1c7 cells were treated with ZER (100 µM) for 1 or 3 hours. HSF1 phosphorylated at Ser326 in cell lysates was detected by ELISA. As a positive control, cells were exposed to heat shock (HS; incubation at 43°C in waterbath) for 15 minutes and examined. This experiment was performed in quadruplicates. (B) Cells were treated with the vehicle, ZER (Z; 10, 25, 50 µM) or geldanamycin (G; 1 µM) for 3 hours, then total RNA was subjected to qRT-PCR to semi-quantify the expressions of HSP90α, HSP90β, HSP70, and HSP40. HPRT expressions were also measured as internal standards. This experiment was performed in triplicates. a, versus CTL by Dunnett's test (<i>P</i><0.05). (C) Cells were treated with the vehicle, ZER (50 µM), or geldanamycin (GEL; 1 µM) for 6-24 hours, then lysed for western blot analysis. (D) Cells were treated with Lipofectamine™ 2000 and a siRNA solution (control and HSF1, 75 nM) for 6 hours. The culture medium was replaced with DMEM containing 10% FBS and incubated for another 24 hours. Cell lysates were subjected to western blot analysis. (E) Cells were treated with Lipofectamine™ 2000 and a siRNA solution (control and HSF1, 75 nM) for 6 hours, then the culture medium was replaced with DMEM containing 10% FBS and incubation was performed for another 24 hours. Then, siRNA-transfected cells were treated with the vehicle, ZER (50 µM), or geldanamycin (GEL; 1 µM) for 15 hours, and total RNA was subjected to qRT-PCR to semi-quantify the expression of HSP70. HPRT expression was also determined as an internal standard. This experiment was performed in quadruplicates. The various characteristics were significantly different, as shown by Tukey-Kramer test result (<i>P</i><0.05). (F) Cells were treated with the vehicle, ZER (50 µM) or tunicamycin (TUN; 1 µM) for 6 hours, then total RNA was subjected to qRT-PCR to semi-quantify the expressions of CHOP and GRP78. HPRT expressions were also measured as internal standards. This experiment was performed in triplicates. *<i>P</i><0.05 vs. DMSO by Dunnett's test.</p

Screening of food ingredients for HSP70 inducing activities.

<p>Hepa1c1c7 cells were treated with the vehicle, nutrients such as all-<i>trans</i> retinol (ATRA; vitamin A), ergocalciferol (VD; vitamin D), α-tocopherol (VE; vitamin E), phylloquinone (VK; vitamin K), thiamin (VB1; vitamin B₁), nicotinic acid (VB3; vitamin B₃), pyridoxine (VB6; vitamin B₆), cyanocobalamin (VB12; vitamin B₁₂), ascorbic acid (VC; vitamin C), folic acid (VM; vitamin M), asparagine (Asn), arginine (Arg), fructose (Fruc), sucrose (Suc), manganese (II) sulfate (Mn), and zinc chloride (Zn) (A) or phytochemicals such as quercetin (QUE), ellagic acid (EA), (-)-epigallocatechin-3-gallate (EGCG), phenethyl isothiocyanate (PEITC), zerumbone (ZER), α-humulene (HUM), curcumin (CUR), and ursolic acid (UA) (B) for 6 hours, then total RNA was subjected to qRT-PCR to semi-quantify HSP70 expression. HPRT expressions were also measured as internal standards. All compounds were treated at their nonlethal and maximum concentrations (Mn: 0.016 µM, VD: 10 µM, VB1, VB3, VB6, Asn, and Arg: 500 µM, PEITC: 2 µM, HUM: 25 µM, all others treated at 50 µM). These experiments were performed in triplicates. *<i>P</i><0.05 vs. DMSO by Dunnett's test (A, B).</p